The non-polar extracts of aromatic medicinal plant species are mainly composed of essential oils (EOs) and other complex mixture of compounds, mainly monoterpenes, sesqiuterpenes, long chain aliphatics, and their oxygenated derivatives (alcohols, aldehydes, esters, ethers, ketones, phenols, and oxides). These constituents may be obtained from different plant materials such as flowers, bark, wood, twigs, leaves, and roots [1
The genus Albizia is comprised of about 150 species widely distributed in tropics, with great diversity in Africa and Central South America [2
]. Albizia adianthifolia
(Schumach), (Family: Fabaceae, Mimosoideae), is a large deciduous tree, trivially known as flat crown Albizia, that grows and habitats well in both dry woodland and sandy soils. It is valued as a shade tree in cocoa and coffee plantations in Sierra Leone as well as for soil conservation. Amongst other economic important uses of this species is its utilization in light constructions (e.g., rafters, posts) and carvings (e.g., spoons, clubs). Extensive literature survey revealed that various parts of A. adianthifolia
have been used for a number of folkloric applications to treat such diseases such as skin and respiratory tract infections, headaches, sinusitis, and malaria, as well as use as analgesic, purgative, anti-inflammatory, and as a psychotic principle [3
genus is comprised of 100 species amongst which is included Pterocarpus angolensis
(DC), (Family: Fabaceae, Papillionoideae). P. angolensis
is mainly found in the tropical regions of Africa as well as the Neotropical regions and Indomalaya [5
]. It is a deciduous large tree, 18–19 m tall with a dark brown bark and a high wide-crowned canopy of shiny compound leaves. The leaves, which are alternate, deep green and imparipinnate, appear at the time of flowering or shortly afterwards. The pod has a diameter of about 2–3 cm, is surrounded by a circular wing which is 8–12 cm in diameter, reminiscent of a brown fried egg, and contains a single seed. This characteristic brown papery and spiky seed pod stays on long after the leaves have fallen from the tree. P. angolensis
grows well on deep sandy soils or well drained rocky slopes and on open woodland areas. Owing to its excellent general properties the timber from P. angolensis
has many applications in the building and furniture industries. P. angolensis
is a woody tree that is revered because of its great natural durability [6
]. The sticky deep red/maroon colored sap is used as dye for fabric. In folk medicine, P. angolensis
is mainly used for gastro-intestinal, urino-genital, fertility problems, respiratory conditions, and skin afflictions [7
]. The traditional use of extracts from A. adianthifolia
and P. angolensis
seem to suggest, among others, efficacy against free radical induced and microbial infections.
There are literature reports on the composition of non-polar constituents from both Albizia and Pterocarpus generra. For example, the GC-MS analysis of the ethanolic and n
-hexane extracts of the stem bark of A. chevalieri
] and the ethanolic extracts of the stem bark and heartwood of P. marsupium
along with their identified (antimicrobial, antioxidant, preservative, lubricant, flavor etc.
) activities were reported [9
]. To the best of our knowledge, no study concerning the non-polar extracts of the heartwood and stem bark of either A. adianthifolia
or P. angolensis
has been reported.
Due to the diversity of folkloric uses of A. adianthifolia
and P. angolensis
against a number of diseases, we considered it worthwhile to determine the non-polar constituents of these two plant species. This was done by GC-MS analysis of the non-polar n
-hexane and chloroform extracts and identification of the components was achieved by comparison of the acquired spectra with the existing NIST library. We were able to isolate other constituents using conventional column chromatography whose structures were elucidated using NMR spectroscopy. The presence of some of these constituents was later confirmed by GC-MS analysis. The GC-MS analysis of the non-polar constituents, not surprisingly, revealed that some components were in fairly high concentrations (major components) while other were only present in trace amounts [10
2. Materials and Methods
Analytical reagents (AR) and general purpose reagents (GPR) solvents were used. The GPR solvents were distilled using simple distillation before use.
2.1. Collection of Plant Materials
The heartwood of A. adianthifolia was collected from Sokoto, north-western Nigeria in January, 2011. The plant species was identified by Mal. Auwal Muhammad, Botany Unit, Biological Sciences, Usmanu Danfodiyo University Sokoto, Nigeria and a voucher specimen, UDUH/ANS/0029, was deposited in the University Herbarium. The stem bark of P. angolensis was collected from Kasane in the northern region of Botswana in June 2012 and was identified at the Botany unit of Biological Sciences, University of Botswana where a voucher specimen was also deposited. The samples were air dried under shade, pulverized into course powder using Thomas Wiley Model 4 and stored in air tight containers until use.
2.2. Extraction Method
The heartwood of A. adianthifolia (657.51 g) was successively and exhaustively extracted with n-hexane, chloroform, methanol, and 10% methanol (aq). The n-hexane and chloroform extracts, upon concentration under reduced pressure using a rotavapor (Buchi, Flawil, Switzerland) (<40 °C), yielded respectively orange-yellow (0.95 g) and yellowish-green (9.16 g) crude extracts. Following a similar extraction protocol the stem bark of P. angolensis (2.21 kg) yielded n-hexane (yellow paste; 51.45 g), and chloroform (reddish-brown paste; 11.6 g) crude extracts.
A small portion of the n-hexane crude extract from each species was dissolved in chloroform and subjected to GC-MS analysis. The main crude chloroform extract from each of the species was separately adsorbed on coarse silica gel (0.2–0.5 mm) ratio (1:1), and allowed to dry before packing or loading onto a column with fine silica gel 60 (0.040–0.063 mm). The extracts were separately eluted under vacuum liquid chromatography (VLC) using n-hexane-chloroform-methanol in increasing (10%) polarity until 100% methanol.
The crude chloroform extract of the heartwood of A. adiantifolia yielded 52 fractions (50 mL each). These fractions were pooled based on the analytical TLC profiling (using acetone/n-hexane, 3:7) to give fractions 1–9 which were encoded ‘’A’’. Fraction ‘’A’’ was then subjected to GC-MS analysis. Using the same procedure, the crude chloroform extract of the stem bark of P. angolensis yielded 79 fractions. These were combined to give sub-fractions A–H. Sub-fraction F was adsorbed on coarse silica gel and subjected to column chromatography using different solvent systems (n-hexane/chloroform/acetone) in increasing (10%) polarity to yield 19 sub-fractions. These were combined which further yielded eight sub-fractions encoded F1–F8. From sub-fraction F3, a white crystalline solid (15.5 mg) was obtained which was identified by NMR spectroscopy as stigmasterol. The presence of stimasterol in the crude chloroform extract was confirmed by GC-MS analysis.
2.3. Procedure for GC-MS Analysis
hexane extracts obtained from the heartwood and stem bark of A. adianthifolia
and P. angolensis
respectively were analyzed separately by GC-MS using a HP-5MS capillary column (30 m × 250 μm, i.d., 0.25 μm film thickness) in an Agilent 6890N gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled to a water GCT Premier mass spectrometer (Waters Corporation, Milford, MA, USA). The carrier gas was helium with a constant flow rate of 3 mL/min. The oven temperature was initially kept at 100 °C for 4 min then ramped at 10 °C/min to 240 °C. The temperature was gradually increased from 8 °C/min to 300 °C and held isothermally for 10 min. An amount of 1.0 μL of the sample (100 ppm in chloroform) solutions was injected in the splitless mode. Mass spectra were obtained by EI at 70 eV over the scan range m
50−800. The compounds were identified by comparison of their mass spectra with those of the NIST 05 L mass spectral library. The spectral match factor limit was set at 700 and any components with match factor less than 700 were not considered [11
2.4. Procedure for Antimicrobial Activity
The antimicrobial activities of the extracts were evaluated using the modified agar overlay method [12
]. The nutrient broth was prepared by dissolving (13 g/L) in distilled water and heating on a hotplate equipped with magnetic stirrer until a homogenous mixture was obtained. Then 50 mL of the nutrient broth was transferred into 250 mL (×5 for each organism) Erlenmeyer flasks and stoppered with cotton wool and aluminum foil. The nutrient agar was prepared by dissolving (28 g/L) in distilled water in a similar manner as the nutrient broth. The two (nutrient broth and nutrient agar) were separately autoclaved for 15 min at 121 °C. The nutrient broth was cooled in a Biohazard cabinet while the nutrient agar was kept in an oven set at 45 °C until ready to use.
Suspensions (10 mL) of the reconstituted pathogens were separately introduced into labeled 250 mL (×5) Erlenmeyer flasks containing 100 mL warm nutrient agar. Using sterile graduated pipettes, the pathogens were administered and spread as evenly as possible onto the pre-coated silica gel TLC plates already loaded with the different compounds in various loadings i.e., 100, 50, 10, 5, 1 (μg). The nutrient agar containing the pathogens administered was allowed to solidify before being incubated for 24 h at 37 °C and 28 °C for the bacterial and fungal strains respectively. The zones of inhibitions (in “mm” after 24 h) were measured after staining of the plates with methylthiazolyltetrazolium chloride (MTT—2 mg/mL). Chloramphenicol and miconazole were used as standards for the bacterial and fungal strains respectively. The entire microbial assay was conducted under strict aseptic conditions.